Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder that is typically fatal within three to five years of the appearance of symptoms. The disease is characterized by the progressive loss of the motor nervous system and voluntary muscle function without great effect on central or sensory neural function. For this reason, afflicted individuals effectively become prisoners within their own bodies before succumbing to respiratory paralysis. To date, there is no truly effective means to slow or reverse the progression of ALS. While motoneuron death is the most recognized characteristic of ALS, the molecular events that underlie ALS are not restricted to the nervous system. A more accurate description of ALS pathology is that paralysis occurs as a result of motor unit loss. In this regard, the fidelity of the fragile connections between motoneurons and skeletal muscle (neuromuscular junctions) is dependent not only on neuronal input but also on the integrity of the effector muscle. Thus, the long-term goal of this research is to understand how skeletal muscle dysfunction contributes to the destabilization of neuromuscular junctions and motoneuron death in multiple forms of ALS. In the defined duration of this proposal, a role for the monomeric G protein Rad (Ras- related Associated with Diabetes) in ALS pathogenesis will be investigated. Rad is of particular interest because: 1) Rad expression is enhanced just prior to presentation of symptoms in muscle of sporadic ALS patients and two established familial ALS mouse models (SOD1G93A and SOD1G86R), 2) Rad is a potent inhibitor of skeletal muscle L-type Ca2+ channels (CaV1.1), and 3) chronic upregulation of Rad causes marked muscle atrophy. Preliminary data also indicate that the two physiological functions of CaV1.1 - L-type Ca2+ channel and voltage-sensor for excitation-contraction (EC) coupling - are progressively impaired in muscle of mice globally expressing SOD1G93A and in mice in which SOD1G93A expression has been restricted to skeletal muscle. Aim 1 will determine whether chronic upregulation of Rad in muscle can cause NMJ destabilization and subsequent motoneuron death. In these experiments, the impact of AAV1-mediated, muscle-specific Rad overexpression on motoneuron viability will be assessed longitudinally using a combination of immunohistological and electrophysiological methods. In Aim 2, a Rad null-SOD1G93A mouse line will be created in order to reveal the direct involvement of Rad in promoting muscle atrophy, NMJ destabilization and motoneuron death. Gross, locomotor, histological, ultrastructural and electrophysiological techniques will then be employed to determine whether genetic ablation of Rad can limit the deleterious effects of SOD1G93A on muscle integrity and motoneuron viability. Using qRT-PCR, immunoblotting and patch-clamp electrophysiology, Aim 3 will test whether enhanced Rad expression and depression of CaV1.1 function are common to multiple forms of ALS.